The present invention relates generally to the use of electric pulses to increase the permeability of a cell and more specifically to a flow-through electroporation system.
Electric fields can be used to create pores in cells without causing permanent damage. This allows for insertion of large molecules into cell cytoplasm. Genes and other molecules such as pharmacological compounds can be incorporated into live cells through a process known as electroporation. The genes or other molecules are mixed with the live cells in a buffer medium. Short pulses of high electric fields are applied to make the cell membranes transiently porous so that the genes or molecules can enter the cells and modify the genome of the cells.
Studies have shown that large size nucleotide sequences (e.g., up to 630 kb) can be introduced into mammalian cells via electroporation (Eanault, et al., Gene (Amsterdam), 144(2):205, 1994; Nucleic Acids Research, 15(3):1311, 1987; Knutson, et al., Anal. Biochem., 164:44, 1987; Gibson, et al., EMBO J., 6(8):2457, 1987; Dower, et al., Genetic Engineering, 12:275, 1990; Mozo, et al., Plant Molecular Biology, 16:917, 1991). However, the efficiency of electroporation, as reflected in the current literature, is usually low (see U.S. Pat. No. 5,019,034, herein incorporated by reference). A typical result is from about 5 to 20 percent transfection depending on conditions, parameters and cell type. Creation of a high efficiency method and apparatus for the of transfer of nucleic acid and the introduction of other preselected molecules into living cells via electroporation is desired.
Genetronics, Inc, San Diego, Calif., has provided an ex vivo flow through electroporation method and chamber in U.S. Pat. Nos. 5,676,646 and 5,545,130, the disclosures of which are incorporated herein by reference.
The present invention provides a method and an apparatus for introducing preselected molecules into a living cell by contacting the cell with the preselected molecules and electrically applying a multiple series of three-step pulses to the cell. The method can be utilized ex vivo.
A three-step pulse process having steps of collection, electroporation, electrophoresis can be used to introduce preselected molecules into the cell. Each three-step pulse includes three discrete electrical impulses each having a specified duration and strength to achieve its respective function.
Each three-step pulse generates an electrical field with a particular field orientation within a flow through chamber apparatus. A rotating electric field can be generated by applying multiple three-step pulses with each three-step pulse having an electric field in a different orientation. This rotating electric field can be configured to introduce transient pores in the living cell without killing the cell. The rotating electric field is provided in a flow through chamber apparatus having more than two electrodes.
A mechanical means of repositioning cells between successive pulses is also provided, e.g., a vibrating table for agitating the cell-molecule mixture. This can increase the areas of permeabiliztion of the living cells.
An apparatus in accordance with the invention can also provide a means to pulse cells at different temperatures and then after pulsing, let the cells recover for a specified residence time at another temperature.
The use of such features provides high viability of cells after electroporation and high transformation efficiency.
These and other aspects and advantages of the invention will become more apparent in light of the following drawings, detailed description and the appended claims.